Protein modification on histone lysines is critical for controlling gene expression, which itself controls the variable and plasfic expression ofthe proteome in diverse cell types. Modifications on lysine are chemically diverse and include acetylation, methylation, ubiquitylafion and sumoylation. We and others have discovered acetyl- and methyl-lysines in many other proteins, and only some directly control gene expression;many are critical regulatory metabolic enzymes. Ubiquitylation controls the life and death of most proteins, and other protein funcfions. The pathways regulating diverse modificafions on lysines are remarkably complex;much remains to be learned. The network of and dynamic interacfions among these modificafion pathways is even more complex;many lysine-modifying proteins are encoded by multi-gene families, have redundant activifies, and multiple substrates, only some of which are known. Cross-talk between modificafions provides an extra layer of regulation. We have developed genefic, protein chip, chemical, microfiuidic and computafional approaches to decrypt and abstract the complex networks defined by these signaling pathways and monitor how they change over time. This proposal extends many unique technologies developed in the last budget period, with a special focus on adapfing these technologies to monitoring dynamic proteomic changes occurring in response to a range of biological sfimuli. These newer approaches are complemented in this Technology Center for Networks and Pathways by applicafion of innovative mass spectrometry technologies, including sensitive and diverse technologies for quantifying dynamics of lysine modification in cells. The yeast metabolic cycle, integrated with cell cycling and DNA integrity is a fascinafing dynamic cycle that will be studied in detail with several ofthe technologies. Diverse Driving Biological Projects centered on lysine acetylation, methylation, ubiquitylafion and SUMOylafion, as well as advanced Training efforts. Including an internship for students in Puerto Rico, are integrated with the Technology Development aspects ofthe proposal. Technologies and resources are acfively disseminated via mulfiple routes;both static and dynamic proteomics datasets will be centrally warehoused/disseminated. RELEVANCE (See instructions): Lysine modification on histones (and beyond) affects fundamental patterns of gene expression and is often deranged in disease states;developing technologies to monitor how they change is essenfial. Because lysine modificafion is so extensively intertwined with human health, aging and disease, the Technology Center for Networks and Pathways could have far-reaching pre-clinical and clinical impact.